2,411,019 Patented Nov. 12, 1946 UNITED STATES mraur OFFICE» 2,411,019 METHOD OF FORMING CARBOXYLIC ACID SUBSTITUTED'AMINES > Frederick C. Bersworth, Verona, N. J. No Drawing. Application May 1, 1943, Serial No. 485,343 ' 5 Claims. (Cl. 260-112) 1‘ . This invention relates to chemistry and more particularly to organic chemistry and has for its object the provision of a method of convert ing the alpha amino acid content of naturally oc curring protein materials, such as casein, al bumin, soy bean meal, beet sugar residues, and protein molecule which consists of a plurality of molecules of glycine ‘ H-CH-COOH NH: " bound together by peptide linkages extending be tween the alpha amino nitrogen group of one glycine molecule and the carboxylic acid group Another object is to provide a method of re of another glycine molecule. This peptide link covering the alpha amino acid content of protein 10 age is generally indicated as follows: materials in the form of carboxylic acid sub —-OC-CH-X stituted amines. NH-oc-cH-x NH~— Still another object is to provide a method of the like, into commercially useful amino com pounds. producing carboxylic acid substituted amines. Other objects will be apparent as the invention is more fully hereinafter disclosed. This application is a continuation-in-part ap plication of my application Serial No. 363,109 ?led October 24, 1940, now issued as Patent (wherein X=H or the remainder of the amino acid attached to the alpha CH group). When this protein molecule is hydrolyzed 11+ and OH- ions enter into the peptide linkages (NH-0C) between adjacent glycine molecules 2,318,046 dated May 4, 1943, which application 20 converting the same to NHz and COOH groups is assigned to the same assignee as the present respectively. In the absence of a basic metal ion application. the NH: and COOH groups of each glycine mole In accordance with these objects I have dis covered that when a protein molecule is hy cule react together'to form an ammonium salt, but in the presence of a basic oxide-0r hydroxide drolyzed in the presence of a basic metal oxide 25 capable of displacing the acid hydrogen, the car or hydroxide, the peptide linkages (—NH—'OC—) boxylic acid group reacts to form a metal salt. between the plurality of alpha amino acid With the NaOH, for example, the sodium salt molecules of which the protein molecule consists, are broken up and that the carboxylic acid group of the resultant alpha amino acids are stabilized 30 by conversion to a metal salt thereby releasing is formed with liberation of H20. the alpha amino nitrogen group for reaction with Other metal oxide and hydroxides, the metal an amino hydrogen of an amino nitrogen group constituent of which is, capable of displacing acid hydrogens, react similarly as will be apparent of an aliphatic amine whereby ammonia (NIH) is liberated with the substitution of the carboxylic 35 to one skilled in the art. However, the more strongly basic metal oxides and hydroxides are acid salt residue of the amino acid onto the preferable as will hereinafter appear. amino nitrogen of the amine resulting in the In the presence of an aliphatic primary or sec formation of a substituted amine. Accordingly, by varying the extent to which the protein mole ondary amine that contains at least one amino cule is degraded or split up into shorter chain 40 nitrogen group having at least one displaceable amino hydrogen which amino nitrogen group is length groups of peptide-linked molecules of the alpha amino acids, a plurality oi’ substituted » attached directly to an aliphatic carbon, the metal amines from the same amine may be obtained , salt of the alpha amino acid reacts therewith with liberation of ammonia (NHa) to form a carbox~ containing carboxylic substituent groups'of vary ing chain length ranging from a chain length 45 ylic acid substituted amine in which the substitu ent carboxylic group for each amino hydrogen of equivalent to that of a single amino acid mole the amine consists of the carboxylic acid salt resi cule up to a chain length equivalent ‘to a large due of the amino acid after the removal there number of amino acid molecules joined together by peptide-linkages with the’end carboxylic acid ' I from of the alphaamino nitrogen group. This re group‘ of the chain stabilized as the metal salt ?oilaction proceeds as follows: of a basic metal and the opposite end group minus ‘ <1) Secondary amine , ‘ the alpha amino group and attached to the amino nitrogen group of the amine. , ' ' The reaction‘ involved in the present invention -' may be most simplyillustrated in the case of a 55 R . H-CH-COONa + N-R-R'= \N-H-CH-COQNB +‘NH; N31. H ‘a? “ ' " 2,411,019 2 H-CH-COON I NH: (2) Primary amine ~ H n-on-coom a) + \\N-R=R-N/ > + ZNHI / H H-CH-COONB (3) Polyamine (iii-amine) NH: ( conditions leads to this conclusion, and it is my belief that the [decompositionof the peptide link age is one of hydrogenation rather than by hy drolysis. i‘ ‘ - , In accordance with the present invention, therefore, it is contemplated subjecting the pro- I tein moleculeto the action of a basic metal oxide or hydroxide in an aqueous solution of‘an ali phatic amine containing at least one amino nitro 10 gen group attached directly to an aliphatic car bon in which group there is at least one displace ~R-N + ma, able amino hydrogen, at a temperature at which \n-on-o'ooNs the reaction proceeds at an economically practi Na00C-CH.H H-CH-COONa NaOOC-CH-H In'the above equations R equals the residue of cal rate, controlling the extent of degradation of an aliphatic amine that contains at least one 15 the protein molecule by regulating the time of the reaction to obtain partial to complete degrada amino nitrogen group, the said residue contain ing an aliphatic carbon bound directly to the tion of the protein molecule with the resultant formation with any given amine and protein long amino nitrogen N; R’ equals a group substituted chain carboxylic substituent groups containing a for one amino hydrogen; R2 equals (CH2)n where n equals at least 2; and in the formula 20 plurality of amino acid molecules bound together by peptide linkages having the carboxylic group > H-CIlH-COONa of the amino acid molecule at one end of the chain neutralized by a basic metal ion and the alpha amino group of the amino acid molecule at the NH: for the amino acid salt, “X” may be present as a substituent group for the H attached to the 25 opposite end displacedwith resultant substitution alpha carbon group (CH) and any other basic of thechain at the alpha CH group of the mole metal ion M may be present in place of Na in cule onto the amino nitrogen of the amine in the carboxyl group. Alanine, for example, " These compounds generally may be indicated by the formula: omen-coon _ place of an amino hydrogen. NH: contains a CHa- group in substitution for the H bound to the alpha CH group present in glycine, and leucine, as another example, con tains the complex group CH: OH-OHa c . in‘ substitution for this same n in glycine. In view thereof, alpha amino acids generally may be written , X-(IJH-COOH NH: wherein X equals hydrogen or a group substituted therefor onto the alpha OH group. Heretofore in the art, hydrolysis of protein in wherein R equals the remainder of an aliphatic amine containing at least one amino nitrogen 45 attached directly to an aliphatic carbon; R’ equals a substituent group for one amino hydrogen; M equals a metal ion; and x equals the remainder of the alpha amino acid molecule attached to the presence of a basic oxide has been effected. the alpha CH group of the said acid. Such hydrolysis procedure results in considerable 50 In the above equations the chain length in oxidation of the hydrolysate product which is un cluded within theparenthesis may consist of n desirable in that decomposition of the complex atoms where n equals zero or any number less structures of which the group X may be com- _ than the total number of atoms included within prised in the formula the protein molecule. As a practical matter, the x- 011-0 0 on NH: ‘ 55 number 11. becomes a number very much less than the total number of amino acid molecules in cluded within a protein molecule, for the reason .is usually effected with resultant loss of valuable that the total, amount of splitting of the plu organic compounds. rality of peptide linkages cannot be controlled or When such hydrolysis, however, is conducted 60 regulated within close limits. However, by vary in the presence of an aliphatic amine, oxidation of the hydrolysates or of the "1!” structure of the alpha amino acids comprising the protein mole cule is substantially eliminated, due, it is believed, ing the time interval of reaction to be short, me dium and prolonged time intervals, substituted carboxylic amines containing substituent groups having a. large number of peptide-linked amino to the fact that the reaction tends to go in the 65 acid groups, a smaller number of peptide-linked direction of metal amide formation as an interme groups and substantially no peptide-linked groups diate reaction product liberating nascent hydro may be obtained. gen for the reduction of the alpha amino group of . As an illustration of the practice of the pres the amino acid. Thereafter when the metal of ent invention, a determined weight of a pro the amide is displaced during the substitution re 70 tein, for example, 500 grams of casein, suspended action, reducing instead of oxidizing conditions in 5000 c. 0. H20 containing 500 grams of di prm are maintained in the solution by the presence 01’ the free metal ion or by its reaction with water to form oxide or hydroxide. This may not be the pyl amine, and 250 grams of a caustic alkali such as NaOH, when heated to a re?uxing tempera ture of about 110° C. under conditions excluding true explanation but the absence of oxidation 75 atmospheric oxygen, for example, in a container 2,411,019 5 . provided with a re?uxing condenser opening, from ' ken. 6 . . Where the time interval of heating is short- ’ the condenser to the air through a one-way'valve , ened to a fraction of this maximum. time‘ inter permitting the escape of uncondensed gases to the atmosphere at a detemiined positive pres sure, will when heated for varying time inter vals rangingfrom 5 hours to 20. hours, produce the sodium salt of substituted carboxylic amines wherein the substituent carboxylic group is of varying chain length decreasing with increase in the time of heating. 10 val, intermediate poly-meric carboxylic substit- I ‘ The amount of the amine added to the above solution is in large excess or that theoretically required to react with the total number of alpha amino acid molecules present in the protein, and the amount of caustic alkali present is in excess 15 uent groups are obtained. _ The protein casein has been reported'as con sistingyin large part, of glutamic acid cans-N112- (000K) 2 However, when treated as above described and 'reacted with an amine to the extent destroying all peptide-linkages the carboxylic acid substitu ent product obtained consists mainly of the acid complex (CH3) 2'CH'CH2-CH' (NHz) COOH, indi cating that the main alpha amino acid‘constitu ent of casein is the acid leucine. ' ~ of the total amount of such alkali required to However, a considerable number of other alpha effect neutralization of all of the carboxylic acid amino acids are present in casein and the mixture groups present in the plurality of alpha amino of tetra carboxylic acid substituted diamines ob acid molecules present in the said protein. The tained by the practice of ‘the above described amount of the excess amine and caustic alkali 20 invention appears to have many valuable prop erties not normally present in a substantially may be varied widely without essential departure from the present invention, as such excess insures pure tetra carboxylic diamine, such as that ob tainable by the reaction of» the substantially pure the progress of thereaction in the direction of ' carboxylic substitution. metal salt of glycine; _ In general, in the above described method of converting the alpha amino acid content of pro tein into substituted carboxylic amines wherein‘ 25 .the substituent group consists of from one to a ‘ ‘ with the diamine. 7 . H-(FH-COONB) NH: , ~ ' The tetra carboxylic acid substituted diamines plurality of alpha amino acid molecules termi nating at one end in a carboxylic salt of a basic 30 formed as above indicated may be recovered from the aqueous solution by acidifying the solution metal and at the other end in a CH group bound to the amino nitrogen of an aliphatic-amine, the amount of water to be employed with any given protein is preferably that which is 'su?icient to maintain a liquid phase reaction between the pro tein and the'amine. Inthe case of amines that are insoluble in the aqueous solution present, I may employ a non-reactive solvent such as a ter tiary alcohol to form an amine solution that is with a strong mineral acid such as HCl or E804 to a pH at which the acid is least soluble and separating the precipitated acid from the metal 35 salt solution. The tetra acid compounds are sub stantially‘insoluble in cold water but are soluble 'in acid and alkali solutions and are generally solubilized by any- metal ‘oxide, hydroxide or carbonate, the metal constituent of which is capa Also in the 40 ble of displacing an acid hydrogen. Many differ ent inorganic and organic compounds may be case of an amine that exists in the vapor state formed from these tetra carboxylic substituted at the temperature of re?uxing. I may feed the miscible with the water solution. amine into the heated solution at a rate which amines for use in a plurality of di?erent ways. The tetra carboxylic substituted diamines in solution. Various ‘other modi?cations of the 45 which the substituent group consists of a chain consisting of a, plurality of peptide-linked alpha practice outlined above may be made without es-‘ amino acid molecules having the carboxylic group sential departure from the present invention as of one end molecules stabilized by a basic metal one skilled in the art will recognize. ion and the opposite end molecule minus its The most useful of the carboxylic acid substi tuted amines producible by the practice of the 50 alpha amino group and connected at the alpha CH group directly to the ‘amino nitrogen of the present invention are‘the tetra carboxylic acid amine, are essentially unstable‘ compounds, sub substituted amines obtainable by reacting an ject to the hydrolytic splitting up of the several aliphatic diamine conforming to the structure peptide linkages contained therein and to further NHz- (CHzin-NHz wherein it may be the numeral, 2, 3, 4, 5, etc., with the protein as above disclosed. 55 substitution therein of an amino nitrogen linkage between the peptide-linked molecules in accord Ethylene diamine, tri methylene diamine, tetra ance with the method disclosed and claimed in methylene diamine,- penta methylene diamine, co-pending application Serial No. 485,344, ?led etc., are typical examples of such diamines. May 1, 1943, which application is assigned to the These tetra carboxylic acid substituted amines have chemical and physical properties that adapt 00 same assignee as the presentapplication, to form long chained resinous—like amino acid substituted the, same for extensive use as de-ionization agents amino complexes of great utility in the art of in water solutions, as extenders and plasticizer forming arti?cial ?bers and threads for the pro agents in association with natural and arti?cial duction of spun and woven products. , polymers of rubber-like structures, and in var ious other ?elds of use, and as intermediates in 65 In the practice of vthe present invention, in place of caustic alkali or in addition to caustic organic synthesis reactions generally. a‘kali, substantially any metal oxide or hydroxide When ethylene diamine, for example, is added whose metal ion is capable of displacing acid to the alkaline solution above described, in an hydrogens from the carboxylic acid group may amount providing one molecule of the diamine for each four (4) molecules of alpha‘amino acid 70 be ‘employed. For example, an alkaline earth metal oxide or hydroxide, or a, heavy metal oxide molecules present in the protein, the mono meric or hydroxide such as iron oxide or hydroxide or tetra‘ carboxylic acid (Na salt) substituted di-. various mixtures of metal oxides and hydroxides amine is obtained where the time of reaction is“ may be employed, if desired. Beryllium oxide extended to the point where the biurette test shows that all the peptide-linkages have been bro 75 and hydroxide and various alkali metal com’ permits complete absorption of the amine in the 9,411,019 7 8 , reconstituted during hydrolysis and the amount pounds with other metal oxides such as plum bates, beryllates, vanadates, molybdates, etc., may alternatively be employed to good advantage in of said organic base being in large excess or that ‘ ' amount providing at least .one displaceable amino the present invention. In general, however, I hydrogen for each alpha amino nitrogen group ' have found it preferable to employ an alkali metal hydroxide as the alkali metal‘salts- of the amino of the neutralized reconstituted- alpha amino acids, the time interval of heating being select acids are not only the most soluble but are also the most stable of the metal salts under the con , the neutralized alpha amino acid hydrolysate ditions of reaction and moreover produce readily product for substitution reaction with said or ed to provide the said desired chain length in soluble salts with strong mineral acids‘ facilitating 10 subsequent precipitation and puri?cation of the l ganic base. . 2. The method of converting the alpha amino ‘ tetra carboxylic substituted diamine. - acid content of protein materials into carboxylic acid substituted amines having a determined In place of casein, any of the naturally occur-_ ring protein materials may be used, such ‘as soy chain length protein hydrolysate substituent bean meal, ‘sugar beet residues, albumin, etc. 15 group therein, which comprises‘ suspending a Theparticular carboxylic substituent group ob tained by the use of these different proteins will, of course, vary with the particular protein em known weight of the protein in‘ water, adding thereto an amount or an inorganic base at least su?icient to neutralize substantially all of the car boxylic acid groups contained in the alpha amino ployed, depending‘ upon ' the particular alpha amino acid or acidsof which the protein molecule 20 acid content of said protein material and an is comprised. The basic reaction resulting in the amount of ‘an aliphatic amine containing at ‘ splitting up of the peptide-linkage with resultant least one amino nitrogen group having at least displacement of the alpha amino nitrogen group one displaceable amino hydrogen therein, said of the amino acid and the :formation of a car amount being in large excess of that amount pro boxylic substituted amine wherein the substituent 25 viding one displaceable hydrogen for each alpha group is the carboxylic residue of the amine acid amino nitrogen group of the alpha amino acids and may consist of a plurality of peptide-linked present in said protein material, and heating molecules of the amino acid, as hereinabove de said suspension to a re?uxing temperature under scribed, however, remain the same. ' a positive pressure of the uncondensed gases The above indicated reaction resulting in the 30 evolved from said suspension su?icient only to formation of carboxylic substituted amines upon prevent the entry of atmospheric gases to the the hydrolysis of alpha amino acid molecules reacting compounds of said suspension, venting , joined together by Peptide-linkages in the pres the excess pressure of the said uncondensed gases ence of a basic metal oxide or hydroxide and in to the atmosphere, for an extended period of time the presence of an aliphatic amine having at 35 providing the said determined chain length pro least one amino group withat least one displace _ tein hydrolysate substituent group. able amino hydrogen, is characteristic of all alpha amino acids existing in molecular form or in chain form as a result of peptide-linkage, such as protein or synthetic peptide-compounds, and 40 insofar as the present invention is concerned peptides and poly peptides of an alpha amino acid, such as the well known di keto piperazine‘ condensation products, are substantial equiva lents for protein. - ' v 3. The method of converting the alpha amino acid content of casein into carboxylic acid sub stituted amines having a mono to poly molecu lar chain length protein hydrolysate substituent group therein, which comprises suspending a, known weight of the casein in water, adding thereto a caustic alkali and an aliphatic amine containing at least one amino nitrogen group 45 having at least one displaceable amino hydrogen Having hereinabove described the present 111-: therein, and heating the suspension to a re?uxing vention generically and speci?cally and given two speci?c examples of the practice of the same it is temperature under a positive pressure of the un condensed gases evolved from the suspension su?icient only to exclude atmospheric gases from believed apparent that the same may be widely varied without essential departure therefrom and 50 the heated suspension, the excess of said uncon densed gases being vented to the atmosphere, the . all such are contemplated as may fall within the amounts of said caustic alkali and said aliphatic scope of the following claims. ‘ _ amine relative to the amount of said casein be ' What I claim is: ing at least sufficient to provide for the complete 1. The method of treating protein materials to convert the alpha amino acid contentvof the 65 neutralization by said caustic alkali of all car boxylic acid groups in the alpha amino acid hy protein into metal salts of carboxylic acid sub drolysate product of the said casein and to pro stituted amines having a desired mono to poly vide a large excess of the amine over that pro molecular chain length protein hydrolysate sub viding‘ at least one displaceable amino hydrogen stituent group therein, which comprises suspend ing the protein in water‘ and hydrolyzing the al 60 for each alpha amino group in the said alpha amino acid hydrolysate product of the said pha amina acid content of the protein in the casein, andv the time interval of heating being presence of an inorganic base and an organic extended to that providing the desired mono to base consisting of an aliphatic amine ‘having at poly molecular chain length of neutralized pro least one amino nitrogen group containing at least one displaceable amino hydrogen therein, - tein hydrolysate substituent group for substitu tion reaction with said amine. by heating the suspension to a re?uxing temper .4. The method of converting- the alpha amino ature under a positive pressure of the uncon acid content of a protein into an alkali metal salt densed gases evolved from said suspension will of a carboxylic acid substituted amine having a cient only to prevent the entry of atmospheric gases to the reacting compounds of said suspen-‘ 70 desired mono to poly molecular chain length sion, the excess pressure of said uncondensed 1 protein hydrolysate substituent group therein, which comprises suspending a known weight of the protein in about ten times its weight of water, of said inorganic base being at least sufficient adding thereto an- amount of caustic alkali‘ap to provide su?icient metal ions to neutralize all carboxylic acid groups of the'alpha amino acids 76 proximating one-half the weight of protein. and gases being vented to the-atmosphere, the amount 2,411,019 9 an amount of an aliphatic amine having at least acid content of casein into an alkali metal salt one amino nitrogen group containing at least one A of a carboxylic acid substituted amine having a desired mono to poly molecular chain length pro tein hydrolysate substituent group therein, which comprises suspending a known weight of casein displaceable amino hydrogen therein‘, providing a large excess of amine over that theoretically re quired to provide one displaceable amino hydro gen for each alpha amino nitrogen group in the alpha amino acid hydrolysate product of said protein, and heating the suspension to a re?ux kali and about its same weight of dipropyl amine, ing temperature under a positive pressure of the and heating the suspension to a re?uxing tem in about ten times its weight of water, adding thereto about one-half its weight of caustic a1 perature approximating 110° C. under a positive pressure of the uncondensed gases evolved from the heated suspension su?icient only to exclude atmospheric gases from the heated suspension, gases in excess of said pressure being vented to venting the excess uncondensed gases to the at the atmosphere, the time interval of said heat ing being selected within the range 5 to 20 hours 15 mosphere, for a time interval within the range 5 to 20 hours providing the said desired chain to provide the desired mono to poly molecular length protein hydrolysate substituent group. chain length protein hydrolysate substituent uncondensed gases evolved from the suspension su?loient only to exclude atmospheric gases from the heated suspension, the said uncondensed group. 6. The method of converting the alpha amino FREDERICK C. BERSWORTH.